Protein Expression Analysis in Microglia

For protein expression analysis in microglia, microglia cells were starved with a mixture of RPMI 1640 and Prigrow III culture media (1:1) supplemented with 0.5% FCS for 1 h prior to treatment with melanoma-conditioned medium (MCM) or with recombinant cytokines. For experiments employing signaling pathway inhibitors or LIF receptor (LIFR) inhibitor, EC359 (HY-120142, MedChemExpress, Monmouth Junction, NJ, USA), microglia cells were starved with the relevant inhibitor (diluted in DMSO) or with DMSO, as control, for 1 h, prior to treatment with MCM.
Protein detection by Western blot was performed as previously described [6 (link)]. Primary Abs against the following proteins were used: JunB, phospho-STAT3 (Tyr705), STAT3, OSM, and anti-β-tubulin (loading control) (Table S1). Horseradish peroxidase-conjugated goat anti-mouse or goat anti-rabbit (1:10,000, Jackson ImmunoResearch Laboratories, West Grove, PA, USA) was used as secondary Abs. The bands were visualized by chemiluminescence ECL reactions (Merck Millipore, Darmstadt, Germany), and band density was quantified by Quantity One^® software v4.6.6 (Bio-Rad Laboratories).